morphologic and pharmacological investigations in the epicatechin gastroprotective effect

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2012, Article ID 708156, 8 pagesdoi:10.1155/2012/708156

Research Article

Morphologic and Pharmacological Investigations inthe Epicatechin Gastroprotective Effect

A. L. Rozza,1 C. A. Hiruma-Lima,2 A. Tanimoto,1 and C. H. Pellizzon1

1 Morphology Department, Biosciences Institute, UNESP-University Estadual Paulista, P.O. Box 510, 18618-970 Botucatu, SP, Brazil2 Physiology Department, Biosciences Institute, UNESP-University Estadual Paulista, 18618-970 Botucatu, SP, Brazil

Correspondence should be addressed to C. H. Pellizzon, [email protected]

Received 26 January 2012; Accepted 29 February 2012

Academic Editor: Jose Luis Rıos

Copyright © 2012 A. L. Rozza et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Previous studies of the gastroprotective activity of plants have highlighted the importance of the polyphenolic compound epicat-echin (EC) in the treatment of gastric ulcers. This paper aimed to evaluate and characterize the gastroprotective mechanism ofaction of EC using male rats. The gastroprotective action of EC was analyzed in gastric ulcers induced by ethanol or indomethacin.The involvement of sulfhydryl (SH) groups, K+

ATP channels, α2 adrenoceptors, gastric antisecretory activity, and the amount ofmucus in the development of gastric ulcers were investigated. The lowest effective dose of EC providing gastroprotective effects was50 mg/kg in the ethanol-induced gastric ulcers and 25 mg/kg in the indomethacin-induced gastric ulcers. The gastroprotection seenupon treatment with EC was significantly decreased in rats pretreated with a SH compound reagent or an α2-receptor antagonist,but not with a K+

ATP channel blocker. Furthermore, oral treatment with EC increased mucus production and decreased H+ secre-tion. Immunohistochemistry demonstrated the involvement of superoxide dismutase (SOD), nitric oxide (NO), and heat shockprotein-70 (HSP-70) in the gastroprotection. These results demonstrate that EC provides gastroprotection through reinforcementof the mucus barrier and neutralization of gastric juice and this protection occurs through the involvement of SH compounds,α2-adrenoceptors, NO, SOD, and HSP-70.

1. Introduction

Gastric ulcers affect thousands of people worldwide and areconsidered a global health problem. The pathophysiology ofgastric ulcers is related to the disequilibrium between harm-ful and protective factors in the gastric mucosa. Agents thatmay initiate the development of an ulcer include the follow-ing: acid and pepsin secretion, Helicobacter pylori infection,poor diet, alcohol ingestion, and the use of nonsteroidal anti-inflammatory drugs (NSAIDs). Protective factors include thefollowing: an intact mucosal barrier, adequate mucus secre-tion and blood flow, cellular regeneration, prostaglandinsecretion, and epidermal growth factor release [1, 2].

The current treatment options for patients suffering fromgastric ulcers include antacids, sucralfate, prostaglandins,muscarinic antagonists, histamine-2 receptor antagonists,and proton pump inhibitors. However, the long-term use ofthese drugs may cause side effects such as hypersensitivity,arrhythmia, impotence, gynecomastia, and hematopoietic

disturbances [3]. Additionally, their use does not necessarilyprevent the recurrence of the disease.

H. pylori infection has been implicated in the pathogen-esis of active and chronic gastritis, peptic ulcer, and gastriccarcinoma [4]. The triple therapy, based on a proton pumpinhibitor combined with clarithromycin and amoxicillinand/or metronidazole, has been the established first-linetherapy over the past years to eradicate H. pylori infections[5–7]. Standard triple therapy started from eradication ratesof more than 90%; however, until now it has experienced asteady decline, decreasing to 70–80% [8]. There are severalreasons for the loss of eradication efficacy, but the mostimportant is the increasing rate of H. pylori resistance toantibiotics [9].

This circumstance evidences the need for new research todiscover substances that would effectively heal gastric ulcerswith fewer side effects. The medicinal plants are attractivesources of new biomolecules, particularly in the developingworld where infectious diseases are endemic and modern

2 Evidence-Based Complementary and Alternative Medicine

health facilities are not always accessible [10]. Althoughmodern medicine may be available in many developingcountries, natural medicines have maintained popularity forhistorical and cultural reasons. Concurrently, many people indeveloped countries have begun to turn to complementary oralternative therapies, including medicinal herbs [11, 12].

Polyphenols, which include flavonoids and tannins, are agroup of phytochemicals that have been intensely researchedand are known to exert beneficial effects on health [13],including experimental gastroprotective action.

Epicatechin (EC, Figure 1), an isomer of catechin, is apolyphenolic compound present in several plant species.Previous studies have shown the gastroprotective activity ofMouriri pusa [14] in which the EC was the main compoundof the extract. Based upon this research, we sought to char-acterize the mechanism of action of EC in gastroprotection.

2. Material and Methods

2.1. Epicatechin: Determination of Doses and Vehicle Used.(−)-Epicatechin (catalog number 855235) was purchasedfrom Sigma Chemical Co. (St. Louis, MO, USA). In ethanoland indomethacin-induced gastric ulcers, the followingdoses were tested: 25, 50, and 75 mg/kg. The lowest effectivedose was used for all subsequent experiments.

Saline +10% absolute ethanol was used as a vehicle, asEC is insoluble in 0.9% saline. To avoid differences betweengroups, all drug compounds were solubilized in this vehicle,including the positive controls.

2.2. Animals. Male Wistar rats (200–250 g) from the CentralAnimal House of UNESP were fed a certified diet with freeaccess to tap water under standard light-dark cycles (12 hdark-12 h light), humidity (60± 1%), and temperature (21±2◦C). All rats were fasted prior to each experiment as treat-ments were orally administered. Additionally, the rats werehoused in cages with raised floors of wide mesh to preventcoprophagy. All experimental protocols followed the recom-mendations of the Canadian Council on Animal Care andwere approved by the UNESP Institutional Animal Care andUse Committee.

2.3. Experimental Assays

2.3.1. Ethanol-Induced Gastric Ulcers. Male Wistar rats thathad been fasted for 24 h were distributed into six groups (n =7). Animals were then orally dosed with vehicle (10 mL/kg),carbenoxolone (100 mg/kg), or EC (25, 50, or 75 mg/kg). Thesixth group (sham) did not receive either drug or vehicle.After 1 hour, the animals received an oral dose of 1 mL ofabsolute ethanol. One hour after ethanol treatment, the ratswere sacrificed and their stomachs were removed. The stom-achs were then opened along the greater curvature andwashed. The flattened stomach samples were scanned andthe ulcer area (mm2) was measured using AVSoft BioViewsoftware. A small fragment of each stomach was collected forglutathione measurement. Stomach samples were collectedfor histological slide preparation and stained with hema-toxylin and eosin (HE) to analyze the morphological and

O

OH

OH

OH

OH

OH

Figure 1: Chemical structure of epicatechin.

histological characteristics, or the slides were used for im-munohistochemical analyses. A microscopic score [15] wasdetermined for the following parameters: epithelial desqua-mation, hemorrhage, glandular damage, and eosinophilicinfiltration, using a scale ranging from 0 to 3 (0: none, 1:mild, 2: moderate, and 3: severe) for each criterion. Thehighest possible score was 12.

Immunohistochemical Analysis. Six slides were used for eachantibody. Each slide was deparaffinized, rehydrated, andimmunostained by the ABC method. Nonspecific reactionswere blocked with H2O2 and goat serum prior to incubationwith a specific antibody. After rinsing in phosphate-bufferedsaline (PBS, 0.01 Mol/L, pH 7.4), the sections were incubatedin secondary antibody (ABC kit, Easypath Erviegas). Thesections were then washed in PBS buffer, the ABC complexwas applied, and the reaction was carried out in a DABsolution (3,3′-diaminobenzidine-tetrahydrochloride) con-taining 0.01% H2O2 in PBS buffer. After immunostaining,the sections were lightly counterstained with hematoxylinand the immunoreactive cells were observed under a Leicamicroscope using Leica QWin Software (Leica, UK). In thecontrol reaction, the slides were processed without the pri-mary antibody or in the absence of all antibodies. The slideswere stained with antibodies for heat-shock protein 70 (HSP-70), superoxide dismutase (SOD), and nitric oxide (NO)(Santa Cruz Biotechnology). The marked area (μm2) in eachslide was measured using AVSoft BioView software.

Determination of Total Glutathione Levels. The total glu-tathione content in the stomach was quantified using therecycling assay [16]. Stomach samples containing ethanol-induced gastric ulcers were thawed and minced, diluted 1 : 20(w/v) in ice-cold 5% (w/v) trichloroacetic acid, and homog-enized. The homogenates were centrifuged at 7000 g for 15minutes at 4◦C. The resulting supernatant was used to quan-tify total glutathione content by reaction with DTNB (5,5′-ditiobis-2-nitrobenzoic acid). Total glutathione was quanti-fied by measuring the absorbance at 412 nm. The results wereexpressed as nmol total glutathione/g tissue.

2.3.2. Involvement of Sulfhydryl (SH) Compounds in Gastro-protection. Rats were distributed into four groups (n = 7).Two groups of rats were intraperitoneally treated with NEM

Evidence-Based Complementary and Alternative Medicine 3

(7 mg/kg), an SH compound blocker. The other two groupswere treated with vehicle (10 mL/kg). One hour later, eithervehicle or EC (50 mg/kg) was orally administered to twogroups each. The ulcers were induced following the ethanol-induced gastric ulcer model and the ulcer area (mm2) wasdetermined with the aid of AVSoft BioView software.

2.3.3. Involvement of K+ATP Channels or Presynaptic α2-Recep-

tors in Gastroprotection. Rats were distributed into six groups(n = 7). Two groups of rats were subjected to intraperitonealtreatment with the following drugs: the K+

ATP channelblocker glibenclamide (3 mg/kg), the α2-receptor antagonistyohimbine (3 mg/kg), or vehicle (10 mL/kg). One hour later,either vehicle or EC (50 mg/kg) was orally administered totwo groups each. The ulcers were induced following theethanol-induced gastric ulcer model and the ulcer area(mm2) was determined with the aid of AVSoft BioViewsoftware.

2.3.4. NSAID- (Indomethacin-) Induced Gastric Ulcers. Therats were distributed into five groups (n = 7). Vehicle(10 mL/kg), cimetidine (100 mg/kg), or EC (25, 50, or75 mg/kg) was orally administered 30 minutes prior to theinduction of gastric lesions by oral administration of theulcerogenic agent indomethacin (100 mg/kg). The animalswere sacrificed 5 h after treatment with indomethacin [17].The stomachs were removed, opened along the greater cur-vature, and then scanned. The ulcer area (mm2) was deter-mined using AVSoft BioView software.

2.3.5. Evaluation of Gastric Juice Parameters. Male rats wererandomly divided into 6 groups (n = 7). Thirty minutes afteroral treatment or immediately after the intraduodenal ad-ministration of a single dose of vehicle (10 mL/kg), cimeti-dine (100 mg/kg), or EC (50 mg/kg), the rats were subjectedto pyloric ligation [18]. Four hours later, the animals weresacrificed, the abdomen opened, and another ligature wasplaced around the esophagus, close to the diaphragm. Thestomach was removed and its contents were drained into agraduated centrifuge tube, which was centrifuged at 2000 gfor 15 minutes. The total acid content of the gastric secretionswas determined by titration to pH 7.0 with 0.01 N NaOHusing a digital burette (E.M., Hirschmann Technicolor,Germany). The total concentration of acid was expressed asmEq/mL/4 h.

2.3.6. Determination of Mucus Adherence to the Gastric Wall.After 24 h of fasting, anesthetized rats (n = 7) were subjectedto longitudinal incisions slightly below the xiphoid apophysisto place a pyloric ligature. Oral administration of vehicle,carbenoxolone (200 mg/kg), or EC (50 mg/kg) was per-formed 1 h before the ligature. After 4 h, the animals weresacrificed and the glandular portion of the stomach wasweighed and immersed in Alcian Blue solution for the mucusquantification procedure. The absorbance was measured in aspectrophotometer at a wavelength of 598 nm, and the resultswere expressed as μg Alcian Blue/g tissue [19].

0

100

200

300

400

500

600

700

Veh

icle

Car

ben

oxol

one

100

mg/

kg

Epi

cate

chin

25 m

g/kg

Epi

cate

chin

50 m

g/kg

Epi

cate

chin

75 m

g/kg

Ulc

er a

rea

(mm

2)

∗∗ ∗∗∗∗

∗∗∗

n.s.

Figure 2: Gastric ulcer area (mm2) of rat stomachs (n = 7) withethanol-induced gastric ulcers after treatment with vehicle, car-benoxolone (100 mg/kg), or epicatechin (25, 50 or 75 mg/kg). Theresults are reported as the mean ± SEM, analyzed by ANOVAfollowed by Dunnett’s test, ∗P < 0.05 and ∗∗P < 0.01. ns: not signi-ficant.

2.4. Statistical Analysis. Parametric data were analyzed usinga one-way analysis of variance (ANOVA) followed byDunnett’s test or Tukey’s test and compared to the vehiclegroup. The results were presented as the mean ± standarderror of the mean (SEM). Nonparametric data (histologyscoring) were analyzed by the Kruskal-Wallis (nonparamet-ric ANOVA) test followed by a Dunn multiple comparisontest. The results were presented as the median (range). Allanalyses were performed using GraphPad InStat software. Avalue of P < 0.05 was considered significant.

3. Results

3.1. Ethanol-Induced Gastric Ulcers

3.1.1. Gastric Ulcer Area. The vehicle group presented severalhemorrhagic bands, with an average ulcer area of 520.41 ±81.46 mm2. The three doses of EC (25, 50, and 75 mg/kg)tested exhibited gastroprotective effects (P < 0.05 for thelowest dose and P < 0.01 for 50 and 75 mg/kg); however, the50 mg/kg dose exhibited the greatest gastroprotective effect(94.83% gastroprotection). According to Tukey’s test, therewas no significant difference between the ulcer areas of thegroups treated with 50 mg/kg or 75 mg/kg; therefore, thedose of 50 mg/kg was used for all subsequent experiments.The ulcer areas (mm2) are represented in Figure 2.

3.1.2. Microscopic Score. There was no microscopic evidenceof eosinophilic infiltration and hemorrhage, but there wasmild desquamation and glandular damage in the grouptreated with 50 mg/kg EC. The HE staining of the ulcers isdisplayed in Figure 3 and the microscopic score is shown inTable 1.

3.1.3. Immunohistochemistry. For HSP-70, SOD, and NO,the immunoreactive areas in the 50 mg/kg EC-treated groupwere statistically larger (P < 0.01) than those in the control


Table 1: Histological scores (0–12) and immunoreactive areas (μm2) for HSP-70, SOD, and NO in rat stomachs (n = 7) with ethanol-induced gastric ulcers after treatment with vehicle, carbenoxolone (100 mg/kg), or epicatechin (50 mg/kg). The scores are represented bymedian (range), analyzed by ANOVA followed by Dunn, and compared to vehicle, ∗∗∗P < 0.001. HSP-70, SOD, and NO are represented bythe mean ± SEM, analyzed by ANOVA followed by Dunnett’s test, and compared to vehicle, ∗∗P < 0.01.

Analysis Vehicle Carbenoxolone Epicatechin

Score 12 (11-12) 4 (2–7)∗∗∗ 3 (2-3)∗∗∗

HSP-70 3718.93± 479.00 1362.30 ± 244.50∗∗ 7861.07 ± 325.92∗∗

SOD 15631.75± 956.50 12933.21± 581.75 26887.53 ± 971.40∗∗

NO 10741.16± 302.40 11892.80± 324.72 13686.50 ± 581.96∗∗

Table 2: Effects of epicatechin (50 mg/kg) on ethanol-induced gastric ulcer area (mm2) in rats (n = 7) pretreated with the SH reagent NEM(7 mg/kg). The results are expressed as the mean ± SEM and analyzed by an unpaired t-test. P < 0.01 compared to the control group, whichwas orally treated with vehicle.

Pretreatment (i.p) Treatment (p.o) Gastric ulcer area (mm2) Percentage of gastroprotection

VehicleVehicle 251.68± 46.59 —

Epicatechin 30.11± 10.61∗∗ 88.04

NEM (7 mg/kg)Vehicle 1157.40± 266.09 —

Epicatechin 558.70± 86.11 51.73

#

(a)

∗

(b)

∗

(c)

Figure 3: Photomicrography of rat stomachs with ethanol-induced gastric ulcers after treatment with (a) vehicle, (b) carbenoxolone(100 mg/kg), or (c) epicatechin (50 mg/kg). HE staining. ∗Indicates epithelial desquamation, #indicates glandular damage, and the arrowindicates a hemorrhage arrows indicate hemorrhage.

group. These data indicate their involvement in the gastro-protective effect of treatment with EC (Table 1).

3.1.4. Determination of Total Glutathione Levels. The treat-ment with EC (50 mg/kg) maintained the total glutathionelevel near that of the vehicle and carbenoxolone groupsafter the total glutathione-depleting ethanol administration(Figure 4). However, the total glutathione level in EC-treatedanimals was lower than that in the sham-treated group.

3.2. Involvement of NP-SH Compounds, K+ATP Channels,

or Presynaptic α2-Receptors in Gastroprotection. In rats pre-treated with NEM (an SH compound reagent), the gastro-protective effect of EC (50 mg/kg) was reversed (Table 2),indicating the involvement of SH compounds in gastro-protection. Rats pretreated with yohimbine (an α2-receptorantagonist) also exhibited a reversal in gastroprotection upontreatment with EC (Table 3), indicating the involvement of

α2-receptors in the protective mechanism of action of EC(50 mg/kg). However, in rats pretreated with glibenclamide(a K+

ATP channel blocker), the gastroprotective effect of EC(50 mg/kg) was maintained (84.14%), indicating that there isno involvement of the K+

ATP channels in the gastroprotectiveeffects of EC (Table 3).

3.3. Indomethacin-Induced Gastric Ulcers. The vehicle-treat-ed group had a large quantity of small petechiae in the stom-ach and a mean ulcer area of 23.97±4.29 mm2. In this model,the most effective dose of EC was 25 mg/kg, which had a67.83% gastroprotective effect (ulcer area 7.71 ± 1.74 mm2,P < 0.01). A dose of 50 mg/kg offered 57.90% gastroprotec-tion (ulcer area 10.09 ± 2.93, P < 0.05) and the highest dose(75 mg/kg) was not gastroprotective (43.87%) (Figure 5).

3.4. Evaluation of the Gastric Juice Parameters. A comparisonof the gastric juice parameters of the rats that were treated


Table 3: Effects of epicatechin (50 mg/kg) on ethanol-induced gastric ulcer area (mm2) in rats (n = 7) pretreated with glibenclamide (K+ATP

channels blocker, 3 mg/kg) or yohimbine (α2-receptor antagonist, 3 mg/kg). The results are expressed as the mean ± SEM and analyzed byan unpaired t-test. ∗P < 0.05 and ∗∗P < 0.01 compared to the control group, which was orally treated with vehicle.

Pretreatment (i.p) Treatment (p.o) Gastric ulcer area (mm2) Percentage of gastroprotection

VehicleVehicle 599.11 ± 98.78 —

Epicatechin 243.33± 61.05∗ 59.39

GlibenclamideVehicle 134.13± 16.03 —

Epicatechin 21.27± 6.00∗∗ 84.14

YohimbineVehicle 602.64± 102.27 —

Epicatechin 1028.81± 171.94 0

Table 4: Effects of cimetidine (100 mg/kg) or epicatechin (50 mg/kg) on gastric juice parameters in rats (n = 7) with pyloric ligation. Theresults are expressed as the mean ± SEM and analyzed by ANOVA followed by Dunnett’s test. ∗P < 0.05 and ∗∗P < 0.01 compared to thevehicle group.

Route Treatment Gastric juice volume (mL) [H+] mequiv/mL/4 h

OralVehicle 1.22± 0.80 8.03± 1.28

Cimetidine 1.17± 0.36 2.49 ± 0.47∗∗

Epicatechin 1.80± 0.55 4.56 ± 0.56∗

IntraduodenalVehicle 3.45± 0.37 7.82± 0.45

Cimetidine 2.08 ± 0.16∗∗ 4.20 ± 0.63∗∗

Epicatechin 1.82 ± 0.15∗∗ 7.64± 1.29

0200400600800

1000120014001600

Sham

Veh

icle

Car

ben

oxol

one

100

mg/

kg

Epi

cate

chin

50 m

g/kg

GSH

(n

g/g) ∗∗∗∗ ∗∗

Figure 4: Glutathione levels (nmol total glutathione/g tissue) of ratstomachs (n = 7) with ethanol-induced gastric ulcers after treat-ment with vehicle, carbenoxolone (100 mg/kg), or epicatechin(50 mg/kg). The results are reported as the mean ± SEM, analyzedby ANOVA followed by Dunnett’s test, compared to sham group,P < 0.01.

with EC (50 mg/kg) administered by oral or intraduodenalroutes demonstrated that the oral treatment was able todiminish the H+ concentration in the gastric juice withoutmodifying its volume. In comparison, the intraduodenaladministration was not able to decrease the H+ concentra-tion, but it did diminish the volume of the gastric juice(Table 4).

3.5. Determination of Mucus Adherence to the Gastric Wall.There was a significant increase in the amount of gastricmucus adhering to the stomach wall in the EC-treated(50 mg/kg) group (2763.07±117.7μg/g, P < 0.01) versus thevehicle-treated group (2211.73± 98.04μg/g) (Figure 6).

Veh

icle

Cim

etid

ine

100

mg/

kg

Epi

cate

chin

25 m

g/kg

Epi

cate

chin

50 m

g/kg

Epi

cate

chin

100

mg/

kg

Ulc

er a

rea

(mm

2)

∗∗

∗∗∗

05

1015202530

Figure 5: Gastric ulcer area (mm2) in rat stomachs (n = 7) withindomethacin-induced gastric ulcers after treatment with vehicle,carbenoxolone (100 mg/kg), or epicatechin (25, 50 or 75 mg/kg).The results are reported as the mean ± SEM, analyzed by ANOVAfollowed by Dunnett’s test, compared to vehicle group, ∗P < 0.05and ∗∗P < 0.01.

4. Discussion

Commonly used therapies for the treatment of gastric ulcersoften fail to completely heal the ulcers and additionallypresent with many side effects. Furthermore, patients aresearching for new and natural methods of healing their dis-eases. Therefore, there has been an increased interest in thesearch for natural products to treat gastric ulcers. The goal ofthis work was to characterize the gastroprotective mechanismof action of epicatechin (EC), which is a polyphenolic com-pound found in several plant species.

Motawi et al. [20] previously demonstrated that an ele-vation in gastric acid secretion, neutrophil infiltration, andalterations in NO production and oxidative stress are mech-anisms that contribute to indomethacin-induced ulceration.


0500

100015002000250030003500

Veh

icle

Car

ben

oxol

one

200

mg/

kg

Epi

cate

chin

50 m

g/kg

Gas

tric

mu

cus

(µg/

g)

∗∗ ∗∗

Figure 6: Quantification of adherent mucus (μg/g of tissue) inthe gastric mucosa of rats treated with vehicle, carbenoxolone(200 mg/kg), or epicatechin (50 mg/kg), analyzed by ANOVA fol-lowed by Dunnett’s test, P < 0.01.

Indomethacin also induces gastric ulcer formation throughthe inhibition of PGE2 synthesis, resulting in a decline inmucus production and subsequent hemorrhagic ulcers [21].In the two lowest doses tested in this study, EC was able toalleviate the effects of indomethacin and prevent gastric ulcerformation. The effect of EC seen in this study was not dose-dependent, which is consistent with previously publishedstudies on polyphenolic compounds [22–25]. This is mostlikely due to the antioxidant effect of lower doses, whileoxidants may be produced at higher doses [26].

Orally administered ethanol causes injury to the gastricmucosa by decreasing local blood flow [27]. It also decreasesthe levels of total glutathione and SH, which are importantgastroprotective factors [28], and depletes the gastric mucus[29] resulting in gastric lesions. In the ethanol-inducedgastric ulcer assay, the EC protective effect at the three dosestested suggests a gastroprotective mechanism that acts di-rectly in the gastric mucosal cells. The intermediate dose(50 mg/kg) was chosen for subsequent assays because itsgastroprotective effect was significantly higher than the effectof the lower dose (25 mg/kg) and was not different from thehighest dose (100 mg/kg).

The mechanisms that could be responsible for the gastro-protective effects were investigated as follows. EndogenousSH compounds are key agents in mucosal protection againstethanol-induced gastric injury [30]. These SH compoundsprovide protective effects through binding free radicalsformed by ethanol treatment and by controlling the produc-tion of mucus [31]. Our results evidence the importance ofthese compounds in the gastroprotective mechanism of EC.Moreover, SH compounds are also involved in the mainte-nance of the mucus disulfide bridges that, if damaged, renderthe mucus more soluble, resulting in a gastric mucosa that ismore susceptible to injuries caused by harmful agents [32].The involvement of this pathway in gastroprotection mayalso explain why the treatment with EC produced a signifi-cant increase in the amount of mucus in the gastric glands.This is an efficient mechanism because the mucus barrierconstitutes the first line of mucosal defense as it decreases

mechanic shock, blocks bacterial access to the epithelium[33], and prevents reverse diffusion of H+ ions [34].

The pyloric ligation model permits the evaluation of theantisecretory actions of an experimental substance for localor systemic activity. This assay induces ulcers by an in-crease in gastric hydrochloric acid secretion, leading to auto-digestion of the gastric mucosa and breakdown of the gastricmucosal barrier. Orally administered EC exhibited antisecre-tory activity by decreasing the H+ concentration in the gas-tric juice without modifying its volume, an effect which didnot occur in the intraduodenally treated rats. These resultslead to the conclusion that EC acts via a local rather than asystemic mechanism. Furthermore, we suggest that thedecrease in H+ concentration in the orally treated rats canalso be explained by the increase in the gastric mucus pro-duction stimulated by EC, given that the mucus barrier isable to neutralize secreted H+.

Gastric cells produce several antioxidants, including SODand endogenous glutathione, which scavenge reactive oxygenspecies (ROS). An excessive generation of ROS enhanceslipid peroxidation and depletes antioxidant enzymes [35].SOD is one of the most effective intracellular enzymatic anti-oxidants, and it acts by catalyzing the dismutation of super-oxide into oxygen and hydrogen peroxide [36]. Total glu-tathione is an important antioxidant that is essential inmaintaining the integrity of the gastric mucosa. It preventsinjuries caused by noxious agents through protection fromfree-radical-induced damage; however, its levels are reducedduring the ulcerative process [37]. The results indicate thatthe gastroprotective effect is not related to the total glu-tathione pathway but to the SOD antioxidant mechanism.The antioxidant potential of EC was reinforced by previousstudies showing that EC was able to prevent lipid peroxi-dation in the rat brain (data not shown). These importantfindings are evidence that the gastroprotective mechanismof EC is related not only to the reinforcement of the mucusbarrier but also to an antioxidant pathway.

The increase in the immunoreactive area for NO andHSP-70 is also an important result. NO has attracted con-siderable attention as a gastric defensive factor. There is sub-stantial evidence that NO, a gas signaling molecule secretedby the gastric epithelial cells, acts by increasing mucosalblood flow, regulating the secretion of mucus and bicarbon-ate, and inhibiting the secretion of gastric juice [38]. It is alsoknown that HSP-70 is induced inside cells exposed to stressoragents. HSP-70 proteins refold or degrade denatured proteinsproduced by harmful agents [39], providing resistance toulceration.

ATP-sensitive potassium channels (K+ATP) provide a gas-

tric defense by inhibiting neutrophil activation and super-oxide production and by enhancing gastric microcirculation[40]. Our data, however, demonstrate that K+

ATP channelsare not involved in the mechanism of action of EC becausethe blockade of these channels with glibenclamide did notreverse the gastroprotective effects of EC.

Our investigation of gastric mucosal protective mecha-nisms was not focused solely on local mucosal processes.Presynaptic α2-adrenoceptors mediate several responses inthe gastrointestinal tract, including antisecretory action and


mucosal protective effects [41], which result in gastroprotec-tion against different types of mucosal damage. The gastro-protection afforded by EC was reversed by yohimbine, anα2-receptor antagonist, suggesting an active role for α2-adrenoceptors in gastroprotection.

5. Conclusion

Taken together, these findings suggest that EC mediates gas-troprotection through a mechanism that includes the rein-forcement of the mucus barrier and the neutralization ofgastric juices. Furthermore, this occurs with the involvementof SH compounds, presynaptic α2-adrenoceptors, SOD, NO,and HSP-70, in addition to antioxidant effects seen upontreatment with this compound.

Acknowledgments

This work was supported by the Fundacao de Amparo aPesquisa do Estado de Sao Paulo (FAPESP 10/08536-9 andFAPESP 08/53798). The authors would like to thank V. M.Souza F. J. Fontana, and M. R. R. Sarzi for technical support(Laboratorio de Histologia Do Departamento de ClınicaMedica FMB UNESP).

References

[1] L. Laine, K. Takeuchi, and A. Tarnawski, “Gastric mucosaldefense and cytoprotection: bench to bedside,” Gastroenterol-ogy, vol. 135, no. 1, pp. 41–60, 2008.

[2] M. G. Repetto and S. F. Llesuy, “Antioxidant properties ofnatural compounds used in popular medicine for gastriculcers,” Brazilian Journal of Medical and Biological Research,vol. 35, no. 5, pp. 523–534, 2002.

[3] F. K. L. Chan and W. K. Leung, “Peptic-ulcer disease,” TheLancet, vol. 360, no. 9337, pp. 933–941, 2002.

[4] M. J. Blaser, “Disappearing microbiota: Helicobacter pyloriprotection against esophageal adenocarcinoma.,” Cancer Pre-vention Research, vol. 1, no. 5, pp. 308–311, 2008.

[5] W. D. Chey and B. C. Y. Wong, “American College of Gastroen-terology guideline on the management of Helicobacter pyloriinfection,” American Journal of Gastroenterology, vol. 102, no.8, pp. 1808–1825, 2007.

[6] K. M. Fock, P. Katelaris, K. Sugano et al., “Second Asia-PacificConsensus Guidelines for Helicobacter pylori infection,” Jour-nal of Gastroenterology and Hepatology, vol. 24, no. 10, pp.1587–1600, 2009.

[7] P. Malfertheiner, F. Megraud, C. O’Morain et al., “Currentconcepts in the management of Helicobacter pylori infection:the Maastricht III Consensus Report,” Gut, vol. 56, no. 6, pp.772–781, 2007.

[8] O. A. Paoluzi, E. Visconti, F. Andrei et al., “Ten and eight-daysequential therapy in comparison to standard triple therapyfor eradicating Helicobacter pylori infection: a randomizedcontrolled study on efficacy and tolerability,” Journal ofClinical Gastroenterology, vol. 44, no. 4, pp. 261–266, 2010.

[9] M. Selgrad and P. Malfertheiner, “Treatment of Helicobacterpylori,” Current Opinion in Gastroenterology, vol. 27, pp. 565–570, 2011.

[10] A. Samie, C. L. Obi, P. O. Bessong, and L. Namrita, “Activityprofiles of fourteen selected medicinal plants from Rural

Venda communities in South Africa against fifteen clinicalbacterial species,” African Journal of Biotechnology, vol. 4, no.12, pp. 1443–1451, 2005.

[11] S. Al-Khalil, “A survey of plants used in Jordanian traditionalmedicine,” International Journal of Pharmacognosy, vol. 33, no.4, pp. 317–323, 1995.

[12] WHO, Monographs on Selected Medicinal Plants, WHO,Geneva, Switzerland, 1999.

[13] A. Gescher, “New perspectives on dietary polyphenols: takingstock, more to come,” Molecular Nutrition & Food Research,vol. 54, pp. S125–S126, 2010.

[14] M. A. Andreo, K. V. R. Ballesteros, C. A. Hiruma-Lima, L. R.Machado da Rocha, A. R. M. Souza Brito, and W. Vilegas,“Effect of Mouriri pusa extracts on experimentally inducedgastric lesions in rodents: role of endogenous sulfhydryls com-pounds and nitric oxide in gastroprotection,” Journal of Ethno-pharmacology, vol. 107, no. 3, pp. 431–441, 2006.

[15] N. Jahovic, G. Erkanli, S. Iseri, S. Arbak, and I. Alican, “Gastricprotection by α-melanocyte-stimulating hormone againstethanol in rats: involvement of somatostatin,” Life Sciences,vol. 80, no. 11, pp. 1040–1045, 2007.

[16] M. E. Anderson, “Determination of glutathione and glu-tathione disulfide in biological samples,” Methods in Enzymol-ogy, vol. 113, pp. 548–555, 1985.

[17] I. Puscas, C. Puscas, M. Coltau et al., “Comparative studyof the safety and efficacy of ebrotidine versus ranitidine andplacebo in the prevention of piroxicam-induced gastroduode-nal lesions,” Arzneimittel-Forschung/Drug Research, vol. 47, no.4, pp. 568–572, 1997.

[18] H. Shay, S. A. Komarov, S. S. Fels, D. Meranze, M. Gruenstein,and H. Siplet, “A simple method for the uniform productionof gastric ulceration in the rat,” Gastroenterology, vol. 5, pp.43–61, 1945.

[19] S. Rafatullah, M. Tariq, M. A. Al-Yahya, J. S. Mossa, and A. M.Ageel, “Evaluation of turmeric (Curcuma longa) for gastric andduodenal antiulcer activity in rats,” Journal of Ethnopharma-cology, vol. 29, no. 1, pp. 25–34, 1990.

[20] T. K. Motawi, H. M. Abd Elgawad, and N. N. Shahin,“Gastroprotective effect of leptin in indomethacin-inducedgastric injury,” Journal of Biomedical Science, vol. 15, no. 3, pp.405–412, 2008.

[21] T. Yoshikawa, Y. Naito, A. Kishi et al., “Role of active oxygen,lipid peroxidation, and antioxidants in the pathogenesis ofgastric mucosal injury induced by indomethacin in rats,” Gut,vol. 34, no. 6, pp. 732–737, 1993.

[22] L. H. Fermin Sanchez De Medina, J. Galvez, J. A. Romero,and A. Zarzuelo, “Effect of Quercitrin on acute and chronicexperimental colitis in the rat,” Journal of Pharmacology andExperimental Therapeutics, vol. 278, no. 2, pp. 771–779, 1996.

[23] M. E. Crespo, J. Galvez, T. Cruz, M. A. Ocete, and A. Zarzuelo,“Anti-inflammatory activity of diosmin and hesperidin in ratcolitis induced by TNBS,” Planta Medica, vol. 65, no. 7, pp.651–653, 1999.

[24] A. N. Garcıa-Argaez, T. O. Ramırez Apan, H. P. Delgado,G. Velazquez, and M. Martınez-Vazquez, “Anti-inflammatoryactivity of coumarins from Decatropis bicolor on TPA earmice model,” Planta Medica, vol. 66, no. 3, pp. 279–281, 2000.

[25] T. Cruz, J. Galvez, E. Crespo, M. A. Ocete, and A. Zarzuelo,“Effects of Silymarin on the acute stage of the trinitrobenzene-sulphonic acid model of rat colitis,” Planta Medica, vol. 67, no.1, pp. 94–96, 2001.

[26] L. C. Di Stasi, D. Camuesco, A. Nieto, W. Vilegas, A.Zarzuelo, and J. Galvez, “Intestinal anti-inflammatory activityof paepalantine, an isocoumarin isolated from the capitula of


Paepalanthus bromelioides, in the trinitrobenzenesulphonicacid model of rat colitis,” Planta Medica, vol. 70, no. 4, pp.315–320, 2004.

[27] S. Siegmund, S. Haas, A. Schneider, and M. V. Singer, “Animalmodels in gastrointestinal alcohol research—a short appraisalof the different models and their results,” Bailliere’s BestPractice and Research in Clinical Gastroenterology, vol. 17, no.4, pp. 519–542, 2003.

[28] P. A. Bafna and R. Balaraman, “Anti-ulcer and antioxidantactivity of DHC-1, a herbal formulation,” Journal of Ethno-pharmacology, vol. 90, no. 1, pp. 123–127, 2004.

[29] T. Al-Howiriny, A. Alsheikh, S. Alqasoumi, M. Al-Yahya, K.ElTahir, and S. Rafatullah, “Protective effect of Origanummajorana L. “Marjoram” on various models of gastric mucosalinjury in rats,” American Journal of Chinese Medicine, vol. 37,no. 3, pp. 531–545, 2009.

[30] S. Szabo and P. Vattay, “Experimental gastric and duodenalulcers: advances in Pathogenesis,” Gastroenterology Clinics ofNorth America, vol. 19, no. 1, pp. 67–85, 1990.

[31] A. S. Salim, “Sulfhydryl-containing agents: new approach tothe problem of refractory peptic ulceration,” Pharmacology,vol. 46, pp. 281–288, 1993.

[32] J. R. Avila, C. Alarcon De La Lastra, M. J. Martın et al., “Roleof endogenous sulphydryls and neutrophil infiltration in thepathogenesis of gastric mucosal injury induced by piroxicamin rats,” Inflammation Research, vol. 45, no. 2, pp. 83–88, 1996.

[33] A. Belley, K. Keller, M. Goettke, and K. Chadee, “Intestinalmucins in colonization and host defense against pathogens,”The American Journal of Tropical Medicine and Hygiene, vol.60, no. 4, pp. 10–15, 1999.

[34] D. Banerjee, S. A. Hassarajani, B. Maity, G. Narayan, S. K.Bandyopadhyay, and S. Chattopadhyay, “Comparative healingproperty of kombucha tea and black tea against indo-methacin-induced gastric ulceration in mice: possible mecha-nism of action,” Food and Function, vol. 1, no. 3, pp. 284–293,2010.

[35] E. Cadirci, H. Suleyman, H. Aksoy et al., “Effects of Onosmaarmeniacum root extract on ethanol-induced oxidative stressin stomach tissue of rats,” Chemico-Biological Interactions, vol.170, no. 1, pp. 40–48, 2007.

[36] G. Aviello, J. M. Canadanovic-Brunet, N. Milic et al., “Potentantioxidant and genoprotective effects of boeravinone G, arotenoid isolated from Boerhaavia diffusa,” PLoS One, vol. 6,no. 5, Article ID e19628, 2011.

[37] C. Y. Li, H. D. Xu, B. T. Zhao, H. I. Chang, and H. I. Rhee,“Gastroprotective effect of cyanidin 3-glucoside on ethanol-induced gastric lesions in rats,” Alcohol, vol. 42, no. 8, pp. 683–687, 2008.

[38] J. Petersson, M. Phillipson, E. A. Jansson, A. Patzak, J. O.Lundberg, and L. Holm, “Dietary nitrate increases gastricmucosal blood flow and mucosal defense,” American Journalof Physiology, vol. 292, no. 3, pp. G718–G724, 2007.

[39] R. I. Morimoto and M. Gabriella Santoro, “Stress-inducibleresponses and heat shock proteins: new pharmacologic targetsfor cytoprotection,” Nature Biotechnology, vol. 16, no. 9, pp.833–838, 1998.

[40] D. A. Campos, A. F. De Lima, S. R. L. Ribeiro et al., “Gas-troprotective effect of a flavone from Lonchocarpus araripensisBenth. (Leguminosae) and the possible mechanism,” Journalof Pharmacy and Pharmacology, vol. 60, no. 3, pp. 391–397,2008.

[41] K. Gyires, K. Mullner, S. Furst, and A. Z. Ronai, “Alpha-2adrenergic and opioid receptor-mediated gastroprotection,”Journal of Physiology Paris, vol. 94, no. 2, pp. 117–121, 2000.

Submit your manuscripts athttp://www.hindawi.com

Stem CellsInternational

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

morphologic and pharmacological investigations in the epicatechin gastroprotective effect

Documents